Anthocyan is a generic term for anthocyanidin, which has a backbone represented by the following formula (I), in combination with anthocyanin, which is a glycoside formed by binding of saccharide to anthocyanidin.
(I) R1R2delphinidinOHOHcyanidinOHHmalvidinOCH3OCH3pelargonidinHHpeonidinOCH3HpetunidinOCH3OH
Examples of anthocyanidin, i.e., an aglycon, include delphinidin, cyanidin, malvidin, pelargonidin, peonidin, and petunidin. Anthocyanin is referred to as anthocyanidin glucoside when, for example, glucose is bound to the anthocyanidin as a glycoside. Saccharide found in anthocyanin includes: monosaccharide such as galactose and arabinose in addition to glucose; and disaccharide such as rutinose and sophorose.
Anthocyans are widely present in nature, and are mainly used as a natural pigment for food or, because of their functionalities, are extensively used for pharmaceuticals, quasi drugs, cosmetics, and the like in Europe. For example, use thereof as a cicatrizant, as disclosed in Japanese Examined Patent Publication (Kokoku) No. 59-53883, or pharmacological properties thereof which are valuable in the treatment of peripheral blood diseases using anthocyanin derived from blueberry, as disclosed in Japanese Laid-open Patent Publication (Kokai) No. 3-81220, have been discovered. In recent years, the functionality of anthocyanin has drawn attention in Japan for uses of anthocyanin other than as a pigment. The present inventors have also found several useful efficacies in anthocyanin of black currant and these are reported in WO 01/01798.
When these anthocyanins having pharmacological properties are used as pharmaceuticals and the like, highly purified ones are required. Heretofore, however, mass production of highly purified anthocyanins has never been realized. Further, while highly purified anthocyanins are preferably crystalline from the viewpoint of stability, hygroscopicity, and the like, mass production of crystalline anthocyanins has likewise not been realized up to now.
Conventionally, anthocyanin compositions for pharmaceuticals are mainly preparations derived from blueberry with an anthocyanin content of 25% by weight or lower. Thus, at least several hundred mg of an anthocyanin preparation had to be administered per dose in order to exhibit its effectiveness, and the consumption of a small amount thereof could not produce pharmacological effects in practice. Accordingly, compositions containing highly purified anthocyanin at high levels have been awaited.
Highly purified anthocyanin was not present because of the following reasons. For example, in the case of blueberry-derived anthocyanin, there are 15 types of anthocyanin components, and the physiochemical properties of these substances are very similar to one another. Thus, the respective flux peaks thereof overlap with one another in purification using a preparative column or the like. Or, separation and purification were impossible because each component was in a very small amount.
In the case of anthocyanin derived from black currant, for example, four components, i.e., cyanidin-3-O-glucoside (hereinafter it is abbreviated to “C3G”), cyanidin-3-O-rutinoside (hereinafter it is abbreviated to “C3R”), delphinidin-3-O-glucoside (hereinafter it is abbreviated to “D3G”), and delphinidin-3-O-rutinoside (hereinafter it is abbreviated to “D3R”), are contained as anthocyanins. As with the blueberry-derived anthocyanin, due to very similar physiochemical properties among the four substances, even mixtures of these four components have very close chromatography peaks. Even if preparative chromatography or centrifugal partition chromatography were performed to obtain purified anthocyanin, mass production thereof was impossible due to extremely deteriorated yield. The quantitative ratio of representative anthocyanins in black currant is as follows: D3G, D3R, C3G, and C3R are respectively present at 12.5%, 47.9%, 4.1%, and 35.5%. Consequently, purification of a large amount of D3G and C3G components, which are contained at low levels, involved further difficultly, and thus a process for mass producing purified anthocyanin has been awaited.
In contrast, anthocyan has been heretofore known to have a drawback in its stability. The present inventors have applied for patent on a process for stabilizing substances containing anthocyanin at high level by adding phytic acid, saccharides, and sugar alcohols as stabilizers (PCT/JP00/09204). However, when a large amount of anthocyanin is used for making preparations, there is no room to contain these additives. Accordingly, more stable physical properties were required and preparations using crystalline anthocyanin, which is physically more stable, were awaited. Thus, conventionally, high purity anthocyanin was organically synthesized for pharmaceutical applications through many steps by, for example, a process for synthesizing delphinidin hydrochloride (anthocyanidin hydrochloride of an aglycon instead of glycoside) as disclosed in Japanese Patent No. 3030509, although mass production thereof from natural products was not realized.
The anthocyanidin hydrochloride produced by the synthesis method is stable under strong acidic conditions, however, it is likely to be degraded as compared to a glycoside in weak acidic to neutral regions. The application range was thus very narrow. Accordingly, production of anthocyanin which was more stable in acidic to neutral regions as crystals was awaited, although mass production of anthocyanin by the organic synthesis process is currently still unavailable.
Features of anthocyanins are listed in the Dictionary of Natural Products (issued by Chapman & Hall, 1994, London). For example, the crystal of D3R has not been heretofore reported, and while the crystal form of D3G hydrochloride has been reported, its melting point is not described. This indicates that mass production thereof was difficult. Similarly, although the melting point and the crystal form of C3R hydrochloride are described, there is no description on the melting point of C3G hydrochloride. This indicates that mass production thereof was also difficult. Up to the present, only a very small amount of purified anthocyanin could be produced, regardless of whether it is crystalline or not. Thus, there was substantially no study which investigated the reactivity of various enzymes to anthocyanin. Especially, there was no description on the reactivity of rhamnosidase to anthocyanin. Accordingly, a process for mass producing highly purified anthocyanin from natural products without using complicated synthesis processes has been awaited. Further, a process for mass producing crystalline anthocyanin salts by crystallizing purified anthocyanin was also awaited.